Difference between revisions of "Part:BBa K343005:Experience"

Latest revision as of 18:23, 5 November 2010

K343005

UV-Vis determination of beta-carotene and retinal production

Both beta-carotene and retinal have unique and characteristic spectres when studied by UV-vis spectrometry. The spectra can be obtained by harvesting beta-carotene- and retinal-producing cells, re-suspending them in acetone and lysing them, which we chose to do by sonication. Afterwards, the cell debris can be pelleted and the supernatant can be examinated.
The acetone suspension of beta-carotene and retinal can then be subjected to spectrophotometry, and the obtained values and spectra can be compared to those of pure beta-carotene or retinal in known concentrations. This method provides both a qualitative answer to whether or not the desired compound is present and a qualitative indication of the concentration in the cells.
In this experiment cells were prepared and harvested according to protocol [http://2010.igem.org/Team:SDU-Denmark/protocols#EX1.1 EX1.1]. This experiment was performed with six different strains of E. coli:

Wild type TOP10
Wild type MG1655
TOP10-pSB1A2-K274210
MG1655-pSB1A2-K274210
TOP10-pSB1A2-K274210/pSB1C3-K343005 (double transformants)
MG1655-pSB1A2-K274210/pSB1C3-K343005 (double transformants)
Both K343005 and K274210 were constitutively expressed.

Since the first experiment showed no or small amounts of product in cells grown to exponential phase the measurements were performed on cells incubated for 20 hours at 37 degrees Celcius. The graphs obtained from the experiment are presented below:

Figure 1: UV-VIS spectre of six E. coli strains: Wild type TOP10, Wild type MG1655, TOP10-pSB1A2-K274210, MG1655-pSB1A2-K274210, TOP10-pSB1A2-K274210/pSB1C3-K343005 (double transformants), MG1655-pSB1A2-K274210/pSB1C3-K343005 (double transformants)

In the spectrum a sudden drop between 330 and 320 nm occurs, this can be caused when the samples was auto zeroed according to acetone which is the solvent used in the extraction of the beta-carotene and Retinal.
The graph also shows that cell material interferes with the UV-vis measurement where the retinal has the strongest absorption. Therefore, an organic separation is required prior to the measurements.
In order to assess the results, standard dilutions of beta-carotene and retinal were made and measured. The concentrations were 1mM, 100 µM, 50 µM, 25 µM, 10 µM, 5 µM, 1 µM, 100 nM and 10 nM. The standard dilutions were measured on the spectrophotometer. The resulting graphs are presented below:

Figure 2: UV-VIS of beta-carotene at different concentrations. The spectre looks reliable

Figure 3:UV-VIS of retinal at different concentrations. The spectre does not look reliable

The spectrum for the Beta-carotene looks normal and reliable, the standards for retinal however doesn’t look reliable. The spectra for the three lowest concentrations appears to be distorted and implies that something is interfering with the measurement.
The measurement jumps rapidly from nearly nothing to the maximum absorbance, this might be because the concentration of retinal is to low to be measured as a spectrum, but the concentration of the individual derivates of retinal might be high enough to be measured.
It might also just be that the concentrations are to low and something else is influencing the measurements.
Because of the error in the UV-vis spectra concerning retinal detection in both the standards and samples the next characterization experiments is preformed on a HPLC.

All data can be seen under Raw data

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